P-hydroxyphenyl propionic acid derivatives as antiproliferative agents

ABSTRACT

Derivatives of the compound p-hydroxyphenyl propionic acid characterised by those derivatives having the general formulas (I) and (Ia), where n can take the values 1,2,3; R can be H or CH 3  and R 1  can be CH 3  or H with pharmacological activity and their application in medicine for the treatment of disorders of the immunological system.

FIELD OF THE TECHNIQUE

[0001] Within the therapeutic group of non-steriod antiinflammatoriespropionic acid derivatives occupy an outstanding place from both thetherapeutic and commercial points of view.

[0002] Within this subgroup we can distinguish first of all ibuprofen(1), which was the first of a series in which we can today find naproxen(2), ketoprofen (3) and fenbufen (4) among others.

[0003] The pharmacodynamic characteristics of all these products issimilar, which they present in varying degrees of antiinflammatory,antithermic, analgesic and antiplatlet activity, all of them beingnon-selective inhibitors of ciclooxygenases (Cox I y Cox II)(Terapéutica farmacológica del dolor. Jesús Flores 1993. Ed. EUNSA.Pamplona 1993. Colección clinica de la salud. Capitulo 5, pag:121-156.).

[0004] The structural analogy between compounds (1) and (VI) (the latterbeing originally found and described from a fern “Asplenium onopteris”)has led us to a pharmacological study of the two series offunctionalised molecules starting from

[0005] in accordance with that shown in diagrams 1 and 2.

[0006] The central idea of the present invention therefore consists ofobtaining a series of molecules on the basis of an adequatefunctionalisation using compound VI of Diagram 1 and using thecorresponding derivatives of the aromatic rings, benzene andnaphthalene, as functionalised syntones.

ANTECEDENTS OF THE INVENTION

[0007] The cell, the structural and functional unit of all livingbeings, is governed by a series of mechanisms that take decisions whichin turn determine different conducts: proliferation, differentiation,activation, senescence and apoptosis. In higher organisms, there aresome tissues in which there exist stem cells that generate thefunctional mature cells by proliferation and differentiation.

[0008] Within the systems of higher organisms, the immunological systemconstitutes an essential defence mechanism for preserving the viabilityof the individual. Multicellular beings, including humans, findthemselves in an environment with a great many microorganisms that canpenetrate into their interior and use them for their own growth. Theimmune system is capable of recognising microorganisms and triggering aneffector response leading to their destruction or functionalcancellation. Moreover, multicellular beings undergo errors in cellproliferation processes and they accumulate mutations that lead to thetumoral transformation of some of their components. The immune system isalso capable of recognising cells that have undergone neoplastictransformation and of successfully suppressing tumoral growth anddissemination. Nevertheless, the effector capacity of the immune systemcan provoke inflammatory tissue alterations with lesions toparenchymatous components. These processes are accompanied byinfiltration and proliferation of cells from the immune system into thetissues. Some originate in the response to infectious agents and can beacute and systemic, such as sepsis and multiorganic inflammatoryresponse, or they can be chronic and localised such as hepatitis,tubercular arthritis, etc. Other inflammatory processes mediated by theimmune system are those known as autoimmune, which are triggered in thepresence of the actual components of the organism, such as rheumatoidarthritis, inflammatory disease of the digestive tract, etc. As acellular system, the immune system can also undergo tumoraltransformations giving rise to malign lymphoproliferative syndromes. Theimmune system can also participate in aptogenis of tissue damage bychronic processes such as some demencias and arteriosclerosis.

[0009] In the analysis of immunological responses, a distinction isdrawn between natural or non-specific immunity and acquired or specificimmunity. The latter is in turn divided into tumoral immunitycharacterised by the production of antibodies by type B lymphocytes andin cell mediation response via T lymphocytes. The immunological responseconsists of a complex feedback network in which autocrine and paracrinemediators, cytokins, growth factors, etc., all play a role, in additionto the mediators responsible for connection with the endocrine systemand the nervous system.

[0010] An essential element in the generation of the specificimmunological response is the capacity to expand lymphocytesubpopulations by antigen stimulus determined via a complex process ofrecognition and processing of the antigen followed by a process ofpresentation to the effector cell that finally generates the response.This process can be summarised by saying that the proliferation of thesubpopulations corresponding to the antigen is the essential property ofmature lymphocytes (Assas A K, Lichtman A H, Pober J S. Cellular andMolecular Immunology. 2nd ed. W.B. USA: Sanders Company, 1994:31-3).

[0011] The cellular mechanisms for proliferation in turn imply a complexmechanism for the reception of signals external to the cell via membranereceptors, and the transmission of those signals to the cell nucleus inorder to put into operation the mitotic mechanisms which likewise implynuclear and cytoplasmatic processes (Metezeau P H, Ronot X, LeLoan-Merliquac Q, Ratinard M H. La Citometrie en Flux. In: Le GordeCellulaire. Paris: MEDSI/Mc Gram Hin, 1988:77-87).

[0012] The physiological mechanisms of the immunological system mustnecessarily include an availability of “defensive” cells at sites wheretheir activity is needed, hence the existence of “call” mechanisms,circulation, recruitment and adhesion.

[0013] The efficiency of the immunological system is neverthelesssubject to dysfunctions which can in general terms be separated intothree fundamental types, as has been stated previously. Proliferativedysfunction, that in which a cell population or subpopulationproliferates out of control, giving rise to various types of leukaemiasand lymphomas and other malign and benign lymphoproliferative syndromes.Functional dysfunction therefore implies an exacerbation of the responsethat gives to, for example, autoimmune pathologies due to errors inantigen recognition, or a decrease in the type of response giving riseto different situations of immunosuppression. One particular caseconsists of pathologies or situations of inflammation (chronic or acute)with the concomitant tissue destruction and functional alteration, whichcan be produced in the context of systemic autoimmune diseases orspecific organs as well as in response to various infectious agents.

[0014] The pathology of the immunological system is in some aspects, andparadoxically so, parallel to the course of technological and socialprocesses. For example, there can be no doubting the close relationbetween the development of the AIDS pandemic with social factors such asthe enormous increase in displacements, the liberalisation of customs orthe unemployment situation with its consequences of margination and drugaddiction (Baiter M, Cohen J. International AIDs Meeting Infects a doseof realism. Science (New Fows) 1998;281:159-60., Mann J M, Tarantola DJ. HIV 1998: the global picture. Sci Am 1998;279(1):82-3., Bartlett J G,Moore R D. Improving HIV therapy. Sci Am 1998;279(1):84-7).

[0015] Another important factor of immunological pathology is thedevelopment of the chemical industry and environmental contaminationwhich propitiates the development of allergies and immunotoxicity(Descotes J. Immunotoxicology of drugs and chemicals. Elsevier Press,1990., Herchman E, Kimber I, Purches IFH. Immunotoxicology: Suppressiveand stimulatory effect of drugs and environmental chemicals on theimmune system. Arch Toxicol 1989;63:257-73).

[0016] Finally, we can mention that, at least in developed countries,the increase in life expectancy, changes in nutrition, modifications tointeractions with infectious agents, the present-day life-style, haveall been associated with a greater incidence and prevalence ofautoimmune disease and autoimmunity with inadequate recognition oftissues, which are seen as “not one's own”, as in rheumatoid arthritis(Lugmano R, Gordon C; Bacon P. Clinical Pharmacology and modificator ofautoimmunity and inflammatorion in rheumatoid disease. Drugs1994;47(2):259-83), autoimmune diabetes (Riestra Moriegue J L, GuerroSilva R, Fernandez Sánchez J A, Balio Hernández J, Rodriguez Pérez A.Revisión de los immunesupresores en el tratamiento de la artritisreumatoide. Inflamación 1993;IV (6):368-81), etc.

[0017] Finally, among the aspects tied to technological andenvironmental development, we can mention the repercussions of exposureto ionising radiations.

[0018] Knowledge of the immunological mechanisms and the growing socialrepercussions of immunological pathologies have, in the last 30 years,led to the development of substances capable of therapeuticallymanipulating the immunological system (immunomodulators).

[0019] In short, we have to understand that immunological dysfunction isalways ambivalent and the development of the suppression of some of thefunctions can occasion the exacerbation of others (and vice versa), andthat this implies a special difficulty in the effective therapeuticmanipulation of the immunological system.

[0020] One of the most surprising pharmacological facts is thestructural disparity of the different types of products specificallyclassified as immunomodulators (Werner G F, Jolle's P. Immunostimulatingagents: what next? A review of their present and potential medicalapplications. Eur J Biochem.1996;242:1-19), even leaving to one sideantiinflammatory substances and cytostatic agents (antimitotics).

[0021] On the basis of these considerations, there is a clearpossibility of designing new active principles provided withpharmacological activity towards the immunological system based on sometype of interference with uncontrolled processes of differentiation,activation, proliferation or programmed cell death (apoptosis).

[0022] One of the essential aspects already mentioned and to which thispresent invention is especially tied is the capacity of one type ofimmune cell, the lymphocytes, to enter into proliferation (mitosis) viadifferent stimuli for which it has specific receptors.

[0023] So, one way of blocking this proliferative phenomenon could be toblock one or several of the activation mechanisms, i.e., the blocking ofspecific receptors, to interference with the transmitters of the signalas far as the cell nucleus.

[0024] The present invention describes the obtaining of a set ofsubstances capable of interfering with different mechanisms for thetransmission of activation signals in immunocompetent cells, therebyblocking processes of cell proliferation. This will be able to permitthe therapeutic use of those substances in processes accompanied by aninadequate proliferation of immune system cells. This implies processesof inflammatory tissue infiltration with lymphocyte proliferation suchas what are known as autoimmune diseases and inadequate responses toinfectious agents with induction of tissue damage mediated by the immunesystem, and evidently also including malign and benignlymphoproliferative processes. The lymphocyte proliferation alsoparticipates pathogenically in various chronic diseases such asamyloidosis, Alzheimer type demencia, arteriosclerosis, etc., whichcould benefit from the use of these agents.

[0025] The design of such substances starts from the presence of3-(4-hdroxyphenyl)propionic acid (II) in methanolic extracts of the fernAsplenium onopteris (Hernández Silva H. Aportación a la fitoquímica dehelechos. Síntesis y funcionalización de una nueva molécula naturalbioactiva. Tesis Doctoral, Universidad de La Laguna (1996)). Thestructural similarity of this molecule to certain non-steriodantiinflammatories led to its use as lead product for itsfunctionalisation and pharmacological evaluation.

[0026] There exist antecedents of pharmacological activity of lactones,in particular cytostatic activity of sesquiterpene lactones (Kupchan SM, Eakin M A, Thomas A M. Tumour inhibitors. 69. Structure-cytotoxicityrelationships among the sesquiterpene lactones. J Med Chem1971;14(12):1147-52., 13., Nakagawa M, Hirota A, Sakai H., IsogaiA.Terrecyclic acid A, a new antibiotic from Aspergillus terreus. I.Taxonomy, production, and chemical and biological properties. J Antibiot1982;35:778-82).

[0027] Another antecedent consists of lactones derived from kava, aplant used in traditional medicine in Indonesia (Lebot V, Levesque J. ElKava

un remedio contra el estrés?. Mundo Científico 1987;178:366-70).

[0028] These products are being tested in a wide field of clinicalsituations such as clinical depressions and myorelaxation, thoughhepatic and blood toxicity has recently been described for this type ofproduct (Jappe V, Framke I, Reinhold D, Gollmick H Sebotropia drugreaction resventing from kava-kava extract therapy. A new entity? J AmAcad Dermatology 1988; 38(1):104-6).

[0029] Antiproliferative products must necessarily act at the level ofcontrolling the cell cycle, which means that it is necessary to have aknowledge of the biochemical mechanisms involved in it. This permits(and will permit) the design of inhibitors with greater safety andspecificity (Morgan D O. Principles of CDK regulation. Nature1995;374:131-4., Edgar B A, Lehner C A. Developmental control of cellcycle regulators: A fly's perspective. Science 1996;374:1646-52).

[0030] The outline has very recently been published of the generalsynthesis of a “library” of protein-kinase inhibitor products with theidea of obtaining therapeutic effects starting from specific blockingsof enzyme systems responsible for the progression of the cell cycle(Gray N, Wodicka L, Thunnissen A, Norman T, Kwon S, Espinoza F H et al..Exploiting chemical libraries, structure and genomics in the search forkinase inhibitors. Science 1998; 281:533-8).

[0031] Various indole derivatives have recently also recently beendiscovered (Garoti L, Roberti M, Rossi T, Castelli M, Malagolis M.Synthesis and antiproliferative activity of 3-substituted 1H indole[3,2-d]-1,2,3-triazin-4(3H)-ones. Eur J Med Chem 1993; 33:43-6.) as havethose of gallic acid (Serrano A, Palacios C, Roy G, Crespón C, Villar M,Nocito M et al. Derivatives of gallic acid induce apoptosis in tumoralcell lines and inhibit lymphocyte proliferation. Arch Bioch Biophysicos1998;330(1):49-54.), containing this pharmacological activity, thoughvia different mechanisms.

[0032] From everything stated above, it clearly emerges that themaladjustment of the transduction paths for signals controlling cellgrowth can lead to the development of tumoral pathologies. Consequently,the possibility exists of a design of drugs aimed specifically atblocking the transduction of signals (whether these be of theprotein-protein type or of the “cascade phosphylation” type (Saltiel AR, Samyer T K. Targeting signal transduction in the discovery ofantiproliferative drugs. Chem and Biology 1996;3(11):887-93.). Finally,mention is made of novel antecedents in relation to antiproliferativesubstances (Babit-Le-Bouteiller C, Jamme M F, David M, Silve S, Lanau C,Dhers C et al. Antiproliferative effects of SR31747A in animal celllines are mediated by inhibition of cholesterol biosynthesis at thesterol isomerase step. Eur J Biochem 1998;256:342-59., Dell C P.Antiproliferative naphthopyrans: biological activity, mechanisticstudies and therapeutic potential. Current Medicinal Chemistry1998;5:179-94., Kamei H, Koide T, Kojima T, Hashimoto Y, Hasegawa M.Inhibition of cell growth in cultures by quinones. Cancer Biotherapy &Radiopharmaceuticals 1998;13(3):185-188., Zafra-Polo M C, Figadere B,Gallardo T, Tormo J R, Cortes D. Natural acetogenins from annonaceae,synthesis and mechanism of action. Phytochemistry 1998;48(7):1087-117.,Cheviron N, Grillon C, Carlier M F, Wdzieczak-Bakala J. Theantiproliferative activity of the tetrapectide acetyl-N-SerAspLysPro, aninhibitor of haematopoietic stem cell proliferation, is not mediated bya thymosin □4-like effect on actin assembly. Cell Prolif1996;29:437-46).

DESCRIPTION OF THE INVENTION

[0033] The compounds of the invention have the following chemicalstructures:

[0034] For the purposes of the general description of the invention, weconsider the synthesis of the precursor (II) or of that corresponding tothe naphthyl series (IIa).

[0035] For this we start from the substances (III), (IV) and (V) as perDiagram 1:

[0036] For the “naphthyl” series, see Diagram 2:

[0037] The precursors would be:

[0038] with all the synthesis steps of Diagram 1 being maintained up tothe obtaining of the end products. (I), where the product (IIa) is thecorresponding precursor of the naphthyl series.

[0039] Described below is the synthesis of the product of the invention(I) when n=1, R=R₁=CH₃; n=1, R=CH₃, R₁=H.

[0040] For this, we have prepared the following compounds:

[0041] 3-(4-Hydroxyphenyl) propionic acid, having the followingstructural formula:

[0042] which can be prepared from commercial 4-hydroxybenzaldehyde, offormula:

[0043] For the preparation of the acetyl derivative of (III), offormula:

[0044] The aldehyde (III) was treated with acetic anhyride [(CH₃—CO)₂O]and pyridine (C₅H₅N).

EXAMPLES OF CARRYING OUT THE INVENTION

[0045] The invention is explained via the following experiments:

Preparation of 3-(4-hydroxyphenyl) propionic acid Acetylation of4-hydroxybenzaldehyde (III)

[0046] 6.0 g (48.18 mmoles) of (III) were dissolved in 1 ml of pyridineand 2 ml of acetic anhydride were added. The mixture was left to rest atroom temperature for 24 h. It was poured onto water (200 ml) and anextraction was performed with diethyl ether (3×100 ml). The etherextracts were washed three times, each time with 100 ml of a solution ofdilute hydrochloric acid (0.5 N) and then with sodium bicarbonate. Theether extracts were dried over anhydrous sodium sulphate and vacuumconcentrated to give an oil 6.3 g. (100% yield) of 4-acetylbenzaldehyde.

Spectroscopic data.- IR ν_(max)(CHCl₃) cm⁻¹: 2830, 2743, 1762, 1702,1601, 1503, 1370, 1300, 1194, 1156, 1013, 911, 860, 830, 781, 711. EMm/z (rel. int.): 164[M]⁺ (4), 122[M-42]⁺ (17), 121[M-43]⁺ (34), 92(20),93(14), 65(100). ¹H RMN (δ, CDCl₃): 2.09(s, 3H, CH ₃CO), 7.05(d,J=8.3Hz, 2H), 7.68(d, J=8.3Hz, 2H), 9.74(s, 1H). ¹³C RMN (δ, CDCl₃):20.84(q), 122.16(d) (intensity for 2CH), 131,42(d) (intensity for 2CH),155.14(s), 168.56(s), 190.92(s).

Preparation of the Methyl Ester of 4-acetylcinnamic acid

[0047] In a two-neck flask containing 250 ml of dry benzene, 2 g wereadded of a suspension of sodium hydride (1.19 g, 49.98 mmoles) in anargon atmosphere and at 0° C. Trimethylphosphone acetate was then slowlyadded (8.24 ml, 49.98 mmoles). When the ilide had formed,4-acetylbenzaldehyde was added (5.47 g, 33.33 mmoles). At the end of onehour, the reaction was complete and the mixture was poured onto asaturated solution of NaCl. It was extracted three times with diethylether and the phases were washed twice with distilled water. It wasdried over anhydrous Na₂SO₄, filtered and the solvent was removed bydistillation. After purification in a column of silica gel, 6.74 g (92%yield) were obtained of a white crystalline product, m.p. 61-62° C.

Spectroscopic data.- IR ν_(max)(CHCl₃) cm⁻¹: 1763, 1712, 1637, 1435,1370, 1204, 1165, 985, 834, 791. EM m/z (rel. int.): 220[M⁺] (9),189[M-OCH₃]⁺ (6), 178[M-OCH₃]⁺ (92), 148[M-72]⁺ (10), 147[M-CH₂CO₂CH₃]⁺(100), 119[M- CO₂CH₃ + COCH₃]⁺ (22), 91(23), 77(5). ¹H RMN (δ, CDCl₃):2.19(s, 3H, —COCH ₃), 3.70(s, 3H, —CO₂ CH ₃), 6.31(d, J=16Hz, 1H,—CH═CH—), 7.03(d, J=8.5Hz, 2H), 7.43(d, J=8.6Hz, 2H,), 7.58(d, J=16Hz,1H, —CH═CH—). ¹³C RMN (δ, CDCl₃): 21.02(q), 51.64(d), 117.83(d),122.05(d), 129.11(d, intensity for 2CH), 131.98(s), 143.65(d),151.99(s), 167.21(s), 169.05(s).

Preparation of the Methyl Ester of 3-(4-acetyl-phenyl) propionic acid

[0048] 6.70 g (30.45 mmoles) of the methyl ester of 4-acetylcinamic acidwere dissolved in 100 ml of ethyl acetate and hydrogenated using 5% Pd/C(1.5 g) as catalyst under H₂, for four hours. It was filtered andconcentrated to give an oil, 6.17 g (99.2% yield) of methyl ester of3-(4-acetylphenyl) propionic acid.

Spectroscopic data.- IR ν_(max)(CHCl₃) cm⁻¹: 3025, 2952, 2852, 1750,1723, 1602, 1507, 1437, 1369, 1369, 1296, 1228, 1194, 1166, 1102, 913,849, 638. EM m/z (rel. int.): 222[M⁺] (6), 180[M-(CHCO₂CH₃ + COCH₃)]⁺(98), 149[M-CH₂CO₂CH₃]⁺ (10), 107[M-(CHCO₂CH₃ + COCH₃)]⁺ (100), 91(5),77(4). ¹H RMN (δ, CDCl₃): 2.25(s, 3H, COCH ₃), 2.60(t, J=7.6Hz, 2H),2.29(t, J=7.7Hz, 2H), 3.64(s, 3H, OCH ₃), 6.98(d, J=8.5Hz, 2H), 7.18(d,J=8.5Hz, 2H). ¹³C RMN (δ, CDCl₃): 20.73(q), 29.96(t), 35.25(t),121.26(d, intensity for 2CH), 128.96(d, intensity for 2CH), 138(s),148.99(s), 169.56(s), 173.13(s).

Preparation de 3-(4-hydroxyphenyl)-1-propanol

[0049] To 3.82 g (17.36 mmoles) of methyl ester of 3-(4-acetylphenyl)propionic acid were added 100 ml of dry tetrahydrofurane. To thissolution were slowly added 1.80 g (47.43 mmoles) of lithium aluminiumhydride (LiAlH₄). The reaction mixture was refluxed in an argonatmosphere for 4 hours. At the end of the reaction, water was carefullyadded, extraction was performed three times with diethyl ether and theresult was vacuum dried over anhydrous sodium sulphate, giving 1.70 g(65% yield) of 3-(4-hydroxyphenyl)-1-propanol. Crystallisation inmethanol gave a white crystalline solid m,.p.=52-53° C.

Spectroscopic data.- IR ν_(max)(CHCl₃) cm⁻¹: 3338, 2924, 2853, 1612,1514, 1460, 1376, 1240, 1037, 824. EM m/z (rel. int.): 152[M⁺] (81),134[M-18]⁺ (39), 121[M-CH₂OH]⁺ (8), 108[M-C₂H₅OH]⁺ (30), 107[M-C₂H₅O]⁺(100), 91(12), 77(7). ¹H RMN (δ, CDCl₃): 1.87(m, 2H, —CH ₂—), 2.65(t,J=7.5Hz, aryl-CH ₂), 3.69(t, J=6.5Hz, 2H, CH ₂O), 6.76(d, J=8Hz, 2H),7.06(d, J=8Hz, 2H). ¹³C RMN (δ, CDCl₃): 29.68(t), 62.3(t), 115.22(d,intensity for 2CH), 129.45(d, intensity for 2CH), 133.68(s), 153.80(s).

Preparation of 3-(4-methoxyphenl)-1-propanol

[0050] 2.26 g (14.87 mmoles) of 3-(4-hydroxyphenyl)-1-propanol weredissolved in dry acetone (20 ml) and potassium carbonate (2.05 g, 14.87mmoles) was added, followed by methyl iodide (MeI) (2.11 g, 14.87mmoles). The mixture was kept at reflux in a water bath at 60-70° C. for48 h. After that period of time, it was diluted with water and theacetone was removed at reduced pressure. Extraction was performed withdiethyl ether, and the result was washed with water, dried over sodiumsulphate, filtered and the solvent was evaporated in a vacuum to give anoil (2.35 g) (94.93% yield) of 3-(4-methoxyphenyl)-1-propanol.Spectroscopic data.- IR ν_(max) (CHCl₃) cm⁻¹: 3360, 2937, 2832, 1604,1578, 1515, 1460, 1260, 1034, 1030, 835. EM m/z (rel. int.): 166 [M⁺](32), 148 [M-18]⁺ (15), 135 [M-31]⁺ (5), 121 [M-C₂H₅O]⁺ (100), 107[M-C₃H₇O]⁺ (5), 91 (25). ¹H RMN (δ, CDCl₃): 1.89 (m, 2H, —CH₂—), 2.66(t, J = 7.8 Hz, 2H, phenyl-CH₂—), 3.67 (t, J = 6.5 Hz, —CH₂O), 3.80 (s,3H, phenyl-OCH₃), 6.84 (d, J = 8.4 Hz, 2H), 7.13 (d, J = 8.4 Hz, 2H).¹³C RMN (δ, CDCl₃): 30.94 (t), 34.23 (t), 55.04 (q), 61.77 (t), 113.60(d), 113.68 (d), 129.12 (d), 129.20 (d), 133.80 (s), 157.52 (s).

Preparation of 3-(4-methoxyphenyl)-propanal

[0051] 2.34 g (14.10 mmoles) of 3-(4-methoxyphenyl)-1-propanol weredissolved in 20 ml of dry dichloromethane and slowly added to asuspension of 4.56 g (21.15 mmoles) of pyridinium chlorochromate in 25ml of dichloromethane. Once the reaction was completed, which wasfollowed by thin layer chromatography, 3 ml of diethyl ether were addedin portions and the mixture was filtered over celite. The ether phasewas washed three times with water and left to dry over anhydrous sodiumsulphate for an entire night. The 3-(4-methoxyphenyl)-propanal waspurified by column chromatography using as eluent ethyl acetate:dichloromethane in increasing polarity, with 1.20 g of an oil beingobtained (52% yield).

Spectroscopic data.- IR ν_(max)(CHCl₃) cm⁻¹: 2840, 2730, 1716, 1600,1580, 1511, 1440, 1175, 1109, 1076, 814. EM m/z (rel. int.): 164[M⁺](23), 121(100), 108(30), 91(25), 78(20), 77(16). ¹H RMN (δ, CDCl₃):2.70(t, J=7.5Hz, 2H, —CH₂—), 2.91 t, J=7.3Hz, 2H, phenyl-CH₂—), 3.79 s,3H, phenyl-OCH₃), 6.84(d, J=8.4Hz, 2H), 7.12(d, J=8.4Hz, 2H), 9.60(s,1H, CHO). ¹³C RMN (δ, 27.11(t), 45.38(t), 55.09(q), 113.75(d),113.84(d), CDCl₃): 129.10(d), 129.15(d), 132.21(s), 157.94(s),201.72(s).

Preparation of 3-(4-hydroxyphenyl) propionic acid

[0052] 0.79 g (5.19 mmoles) of 3-(4-hydroxyphenyl)-1-propanal weredissolved in acetone (20 ml) and Jones reagent (chromic anhydride insulphuric acid) (8 ml) was added to it drop by drop until theyellow-brown colour persisted. The solution was stirred for one hour at0° C. The excess reagent was destroyed with methanol and the solutionwas filtered and evaporated at reduced pressure. The residue wasextracted in ethyl acetate, dried over anhydrous sodium sulphate andconcentrated, diving an impure residue (0.6470 g), which waschromatographed over silica gel, obtaining 0.545 g (63.1% yield) of3-(4-hydroxyphenyl) propionic acid. Recrystallisation in methanol gavem.p.=121° C.

Spectroscopic data.- IR ν_(max)(CHCl₃) cm⁻¹: 3400, 2930, 1702, 1597,1511, 1460, 1377, 1298, 1222, 1176, 1105, 919, 928, 774, 722. EM m/z(rel. int.): 166[M⁺] (46), 107[M-59]⁺ (100), 91(6), 77(7). ¹H RMN (δ,2.53(t, J=7.5Hz, 2H), 2.80(t, J=7.5Hz, 2H), CDCl₃): 6.73(d, J=8.4Hz,2H), 7.06(d, J=8.4Hz, 2H). ¹³C RMN (δ, 30.57(t), 36.33(t), 115.93(d,intensity for 2CH), CDCl₃): 130.03(d, intensity for 2CH), 132.53(s),156.50(s), 174.18(s).

Preparation of Methyl 3-hydroxy-2-methylidene-butanoate

[0053] 8.8 g (0.2 ml, 11.3 moles) of recently distilled acetaldehydewere made to react with 25.83 g (27.0 ml, 0.30 moles) of methyl acrylateand 2.5 g (0.002 moles) of 1,4-diazabicyclo[2,2,2]-octane (DABCO). Themixture was left to react at room temperature with stirring, until allthe acetaldehyde had reacted (approximately seven days). At the end ofthat time, extraction was performed with diethyl ether (2×100 ml) andthe result was washed with water (2×400 ml). The ether extracts weredried over anhydrous sodium sulphate and vacuum concentrated, giving23.4 g (90% yield) of a colourless oil of methyl3-hydroxy-2-methylidene-butanoate.

Spectroscopic data.- IR ν_(max)(CHCl₃) cm⁻¹: 3446, 2976, 2855, 1716,1629, 1438, 1282, 1196, 1163, 1094, 1041, 957, 925, 821. EM m/z (rel.int.): 130[M⁺] (1), 115[M-15]⁺ (74), 112[M-18]⁺ (8), 99[M-OCH₃]⁺ (25),98[M-CH₃OH]⁺ (37), 71[M-59(CO₂CH₃)]⁺ (32), 55(100). ¹H RMN (δ, 1.27(d,J=6.8Hz, 3H, CH ₃—), 3.26(s wide, CDCl₃): 1H, OH), 3.68(s, 3H, CH ₃O—),4.55(q, J=6.3Hz, 1H, HC—OH), 5.79(s wide, 1H, CH ₂═C—), 6.13(s wide, 1H,CH ₂═C—). ¹³C RMN (δ, 21.75(q), 51.69(q), 66.63(d), 123.87(t),143.60(s), CDCl₃): 166.91(s).

Preparation of Methyl 2-bromomethyl-2-butenoate

[0054] 8.0 g (46 mmoles) of N-bromosuccinimide were dissolved in 60 mlof dry dichloromethane and were cooled to 0° C. To the mixture wereadded drop by drop 4 ml (50 mmoles) of dimethyl sulphide dissolved in 40ml of chloromethane. The mixture was stirred for 10 minutes at 0° C. Atthe end of that time 5.46 (42 mmoles) of a solution of methyl3-hydroxy-2-methylidene-butanoate in 40 ml of dichloromethane wereslowly added to it. The resulting suspension was stirred for 24 hours atroom temperature until a transparent solution was obtained. To thismixture was added n-hexane (100 ml) and it was poured into a separatingfunnel containing 200 ml of a saturated solution of sodium chloride andice. The organic phase was washed with 100 ml of a saturated solution ofsodium chloride. The aqueous phases were extracted with diethyl ether(2×100 ml), they were washed with water (3×100 ml) and they were driedover anhydrous sodium sulphate. They were vacuum concentrated giving7.45 g (92% yield) of a syrupy liquid of methyl2-bromomethyl-2-butanoate.

Spectroscopic data.- IR ν_(max)(CHCl₃) cm⁻¹: 1715, 1646, 1435, 1284,1194, 1165, 1083, 1049, 876, 766. EM m/z (rel. int.): 194(5), 193[M⁺](5), 163(7), 161[M-OCH₃]⁺ (6), 135(4), 133[M-CO₂CH₃]⁺ (4), 113[M-Br]⁺(95), 81(53), 59(97), 53(100). ¹H RMN (δ, 1.84(d, J=6.7Hz, 3H, H ₃CCH═),3.66(s, 3H, CDCl₃): OCH ₃), 4.14(s, 2H, —CH ₂Br), 6.97(q, J=7Hz, 1H,HC═H). ¹³C RMN (δ, 14.36(q), 23.78(t), 51.77(q), 129.95(s), 143.04(d),CDCl₃): 165.55(s).

Preparation of5-[2-(4-methoxyphenyl-ethyl]-4-methyl-3-methylene-dihydrofuran-2-one (I)

[0055] To a suspension of metallic tin (1.34 g, 11.30 mmoles) in diethylether (22.6 ml) and water (5.65 ml), 2.18 g (11.30 mmoles) of methyl2-bromomethyl-2-butenoate, 2.04 g (12.44 mmoles) of 3-(4-methoxyphenyl)propanal and sufficient quantities of acetic acid were added withstirring. The mixture was refluxed with stirring for 9 hours at 50° C.At the end of that time, water was added and an extraction was performedwith diethyl ether (3×100 ml), it was washed with water and dried overanhydrous sodium sulphate. The result was concentrated obtaining 6.42 gof an oily product which, by means of later treatment with catalyticquantities of p-toluensulphonic acid in benzene at room temperature for12 h, gave 7.92 g (69.5% yield) of5-[2-(4-methoxyphenyl-ethyl]-4-methyl-3-methylene -dihydrofuran-2-one[(I), n=1, R=H; R₁=CH₃] in oil form.

Spectroscopic data.- IR ν_(max)(CHCl₃) cm⁻¹: 2938, 2837, 1736, 1663,1611, 1513, 1456, 1246, 1178, 1124, 1034, 948, 832. EM m/z (rel. int.):246[M⁺] (99), 147[M-(C₅H₇O)]⁺ (98), 135[M-(C₆H₇O₂)]⁺ (19), 134[M-112]⁺(29), 121[M-(C₇H₉O₂)]⁺ (100), 107[M-(C₈H₁₁O₂)]⁺ (14), 92(29), 91(84). ¹HRMN (δ, 1.13(d, J=7Hz, 3H, CH ₃—), 1.74(t, J=7.8Hz, 2H, CDCl₃): CH ₂),2.67(m, 2H, O—CH₂—), 3.17(deformed quartet, 1H, CH), 3.81(s, 3H, O—OCH₃), 4.46(quartet, J=5.8Hz, 1H, HCO), 5.57(d, J=2.5Hz, 1H, ═CH ₂),6.25(d, J=2.5Hz, 1H, ═CH ₂), 6.88(d, J=8.6Hz, 2H), 7.16(d, J=8.6Hz, 2H).¹³C RMN (δ, 13.61(q), 30.59(t), 32.64(t), 37.14(d), 55.00(q), CDCl₃):79.82(d), 113.72(d, intensity for 2CH), 120.52(t), 129.25(d, intensityfor 2CH), 132.68(s), 140.62(s), 157.81(s), 170.30(s).

Preparation of Methyl 3-hydroxy-2-methylidene-propanoate

[0056] To a mixture containing 27 ml (25.83 g, 0.30 mmoles of methylacrylate and 2.5 g (22.29 mmoles) of 1,4-diazabicyclo[2,2,2]-octane(DABCO), formaldehyde was added (generated by pyrolysis ofparaformaldehyde at 200° C.) in a nitrogen current for 1 h. The mixturewas then left to react for 7 days at room temperature. At the end ofthat time an extraction was performed with diethyl ether (2×100 ml) andthe result was washed with water (2×400 ml). The ether extracts weredried over anhydrous sodium sulphate, filtered and vacuum concentrated,giving 19.5 g (77% yield) of methyl 3-hydroxy-2-methylidene-propanoatein oil form.

Spectroscopic data.- IR ν_(max)(CHCl₃) cm⁻¹: 3428, 3009, 2953, 1718,1636, 1513, 1439, 1309, 1272, 1159, 1057, 953, 819. EM m/z (rel. int.):115[M⁺-1] (2), 101[M-15]⁺ (3), 98[M-18]⁺ (2), 85[M-OCH₃]⁺ (89),84[M-CH₃OH]⁺ (73), 83[M-15-18]⁺ (69), 61(12), 55(100). ¹H RMN (δ,3.68(s, 3H, CH ₃O), 4.22(s, 2H, CH ₂O), 5.78(s, CDCl₃): 1H, CH₂═C—),6.17(s, 1H, CH₂═C—). ¹³C RMN (δ, 51.82(q), 61.69(t), 125.50(t),139.39(s), CDCl₃): 166.77(s).

Preparation of Methyl 2-bromomethyl-2-propenoate

[0057] To a solution of N-bromosuccinimide (5.17 g, 26.8 mmoles) in drydichloromethane (40 ml) were added dimethyl sulphide (4 ml) indichloromethane (50 ml), drop by drop with stirring at 0° C. for 10 min.To the resulting mixture was added methyl3-hydroxy-2-metylidene-propanoate (3.15 g, 31.50 mmoles) dissolved indichloromethane (40 ml), leaving it for 24 hours at room temperature. Atthe end of that time, it was poured into an aqueous solution of sodiumchloride and ice. An extraction was performed with diethyl ether (3×100ml), and the result was washed with water and dried over anhydroussodium sulphate. Following vacuum concentration, 4.33 g of a yellow oilwas obtained (89% yield) of methyl 2-bromomethyl-2-propenoate.

Spectroscopic data.- IR υ_(max) (CHCl₃) cm⁻¹: 2997, 2954, 2835, 1736,1612, 1584, 1513, 1437, 1299, 1248, 1176, 1128, 1035, 960, 825, 772. EMm/z (rel. int.): 181, 179[M⁺] (22), 164[M-15]⁺ (20), 1488[M-OCH₃]⁺(5),121[M-CO₂CH₃]⁺ (93), 85[M-CH₂Br]⁺ (13), 61(100). ¹H RMN (δ, CDCl₃):3.76(s wide, 3H, CH₃O), 4.07(m, 2H, CH₂Br), 5.90(s wide, 1H, HC═C),6.25(s wide, 1H, HC═C). ¹³C RMN (δ, CDCl₃): 33.95(t), 53.59(q),129.19(19 (t), 137.27(s), 168.20(s).

Preparation of5-[2-(4-methoxyphenyl)-ethyl]-3-methylene-dihydrofuran-2-one

[0058] To a suspension formed from metallic tin (730 mg, 6.15 mmoles),diethyl ether (12.5 ml) and water (3.1 ml) were added 1.10 g (6.15mmoles) of methyl 2-bromomethyl-2-propenoate and 1.11 g (6.77 mmoles) of3-(4-methoxyphenyl) propanal, ewith catalytic quantities of acetic acid.The mixture was kept warm at 50° C. with stirring for 9 hours. At theend of this time it was poured into water (200 ml) and an extraction wasperformed with diethyl ether (3×100 ml). The result was dried overanhydrous sodium sulphate and vacuum concentrated, giving an oil 780 mg(70% yield) of5-[2-(4-methoxyphenyl)-ethyl]-3-metylene-dihydrofuran-2-one [(I), R=CH₃;R₁=H]. (I)

Spectroscopic data.- IR υ_(max) (CHCl₃)cm⁻¹: 2996, 2926, 2854, 1760,1665, 1612, 1584, 1513, 1464, 1398, 1352, 1300, 1279, 1247, 1178, 1129,1035, 983, 886. EM m/z (rel. int.): 232[M⁺] (23), 147[M-C₄H₅O₂]⁺ (31),135[M-C₅H₅O₂]^(+ (6),) 121[M-C₆H₇O₂]⁺(100), 91(27), 77(27), 65(13). ¹HRMN (δ, CDCl₃): 1,96(m, 2H, CH₂), 2.70(, 2H, —CH₂—), 3.78(s, 3H,O—OCH₃), 4.48(m, 1H, —CHO—), 5.62(d, J=5.0 Hz, 1H), 6.22(d, J=5.0 Hz,1H), 6.83(d, 1=8.5 Hz, 2H), 7.11(d, J=8.5 Hz, 2H). ¹³C RMN (δ, CDCl₃):33.23(t), 33.35(t), 38.13(t), 55.10(q), 76.40(d), 113.61(d, intensityfor 2CH), 121(t), 129.23(d, intensity for 2CH), 132.48(s), 157.89(s),170.18(s).

[0059] Biological Activity

[0060] Biological activities are described of the product described inexample 1 (henceforth referred to as “lactone”).

[0061] Proliferation of Murine Splenocytes. Material and Methods:

[0062] Test of Lymphocyte Proliferation.

[0063] Materials: Processing of the spleen: Fine curved forceps Broadflat forceps Separator forceps Fine scissors Plastic Petri dishes ofFine wire mesh 60 × 15 mm (FLOW) Pipettes of 5 ml Pasteur pipettes 10 mlplastic tubes for Filter paper centrifuging Rubber support fordissection 70% ethanol Hank's balanced solution (HBSS) (FLOW)

[0064] Test:

[0065] Culture medium: DMEM+penicillin 50 UI/ml+streptomycin 50□g/ml+glutamine 2 mM+10% foetal calf serum (complete medium) (FLOW).

[0066] 2 Mercaptoethanol (2ME) (Sigma).

[0067] Mitogenous controls: Concanavalin A (Con A) (SIGMA)Phytohaemaglutinin (PHA) (SIGMA) Pokeweed (PWM) (SIGMA)Lipopolysaccharide 055:B5 (LPS) (SIGMA)

[0068] Tripan blue

[0069] Dishes with 96 flat-bottom wells (FLOW)

[0070] Pipette tips (NUNC)

[0071] Tritiated thymidine (AMERSHAM)

[0072] Wathman filter papers (Titerted/Skatrom)

[0073] Apparatus:

[0074] Refrigerated centrifuge (BECKMAN)

[0075] Liquid scintillation counter LKB 1211 Rack-beta

[0076] Optical microscope

[0077] Skatron Harvester

[0078] Animals:

[0079] Balb/c mice were used, male, aged 6-8 weeks (IFFA-CREDO).

[0080] Method:

[0081] Processing of the spleen:

[0082] The spleen is extracted under sterile conditions

[0083] It is washed with 5 ml of HBSS in a Petri dish

[0084] The spleen is laid on wire mesh

[0085] It is disintegrated with the aid of broad forceps

[0086] The mesh is washed with 5 ml of HBSS and placed in a Petri dish

[0087] The tissue pulp is collected with a Pasteur pipette and taken toa sterile centrifuge tube of 10 ml

[0088] It is washed twice in 10 ml of HBSS and centrifuged at 1,200r.p.m.

[0089] It is resuspended in 11 ml de HBSS

[0090] It is left to rest for 5 minutes

[0091] 10.5 ml are transferred to another centrifuge tube of 10 ml

[0092] A variables count is conducted with tripan blue

[0093] The cell suspension is centrifuged at 1,200 r.p.m.

[0094] The cell “pellet” is resuspended in complete medium at aconcentration of 4×10⁶ cells/ml

[0095] Test:

[0096] In a dish with 96 flat-bottom wells, seriated dilutions are madeof the mitogen problem sample with or without “lactone” in a volume of10 μl in complete medium to which 5×10⁵ M 2ME have been added. All thedeterminations are made in triplicate.

[0097] 100 μl of the cell suspension are then added, leaving a finalvolume of 200 μl.

[0098] The dish is incubated in a stove at 37° C., 5% CO₂ for 66 h.

[0099] Following that incubation period, 1 μCi of tritiated thymidine isadded to each well.

[0100] The dish is again incubated for 6 h.

[0101] Once that new incubation period has ended, the dish is processedin a Skatron Harvester, using special Wathman filter paper for this.

[0102] The filter is dried in air.

[0103] The filters are distributed in β counter tubes to which have beenadded 2 ml of scintillation liquid.

[0104] Each tube is introduced into a vial of 12 ml.

[0105] The cpm are determined in a liquid scintillation counter.

[0106] Controls:

[0107] Negative control: cells in the presence of complete culturemedium.

[0108] Positive control: cells in the presence of some mitogen.

[0109] Results:

[0110] These are expressed as:

[0111] (1) Arithmetic mean of cpm±standard deviation $\begin{matrix}{{{Stimulation}\quad {index}} = \frac{{mean}\quad {cpm}\quad {of}\quad {sample}}{{mean}\quad {cpm}\quad {of}\quad {control}\quad {medium}}} & (2)\end{matrix}$

[0112] The statistical significance is determined by the Student tmethod.

[0113] Tests on Murine Cells. Results:

[0114] A study is first made of the “lactone” effect per se in relationto possible proliferative effects on murine splenocytes. No effect isfound in the range 100-0.75 μg/ml (the sample was dissolved in DMSO at40.0 mg/ml). Nor was are cytotoxicity effects found in the statedranges. TABLE 1 Proliferative response of murine splenocytes following72 h of incubation. Proliferation of murine splenocytes Mitogen (cpm ±DS) Lactone Con A PHA a CD3 LPS μg/ml 2.5 μg/ml 50 μg/ml 20 μg/ml 10μg/ml 100    147 ± 10 19,500 ±  285 ± 20  614 ± 35 2,000 50    190 ± 1019,000 ± 1,235 ± 200 2,700 ± 120 1,800 25    244 ± 15 21,000 ± 5,600 ±300 3,200 ± 400 2,000 12    370 ± 20 6   78,200 ± 725 3   92,600 ± 1001.5 150,400 ±    12,500 0.75 131,523 ±    10,000 0 150,000 ±    20,000 ±40,000 ±      30,000 ± 1,600 15,000 2,000 4,000

[0115] Nevertheless, the lactone clearly demonstrated its capacity forinhibiting proliferation induced by various mitogenic agents: ConA,antiCD₃ and LPS, but not that induced by PHA.

[0116] The results are summarised in the following tables (the cpm arestated following a pulse of tritiated thymidine. The samples ofsplenocytes were cultivated for 72 h in the presence of mitogen andlactone at the stated concentrations).

[0117] The results indicate the capacity of lactone to specificallyinhibit some action signals and not others while in the studiesconducted on murine splenocytes, it is seen how the action indicated bythe lectin of Phaseulis vulgaris (PHA) is not inhibited.

[0118] Tests on Human Peripheral Blood Mononuclear Cells. Material andMethods:

[0119] In order to study more deeply the effects of lactone on cellproliferation, we checked whether the effects described in the abovesection on cells of murine origin were specific to the cell type and thespecies or whether, on the other hand, these effects werexeno-independent. For this, we purified human peripheral bloodmononuclear cells from healthy controls and we proceeded to study thepossible effects of inhibition on the proliferative activity ofperipheral blood mononuclear cells (CMSP) from humans, induced bydifferent mitogenic agents acting alone and/or in costimulation withother mitogens and in the presence or absence of recombinant humaninterleukin 2 (rhIL-2).

[0120] Test on Lymphocyte Proliferation

[0121] Materials:

[0122] Apparatus:

[0123] Air pump for pipetting, Pipetus (Flow Lab., Germany).

[0124] Neubauer counting chamber (Saaringia, Germany).

[0125] Gelaire TC 48 vertical laminar flow chamber (Flows labs.,Germany).

[0126] GPR refrigerated centrifuge (Beckman, United Kingdom).

[0127] SKATRON AS culture collector (Flow Lab. Lierbayen, Norway).

[0128] −30° C. freezer (Selecta, Tarrasa, Spain).

[0129] −70° C. freezer (Selecta, Tarrasa, Spain).

[0130] Beta counter. Betamatic (Kontron).

[0131] Eppendorf multipipette 4780 (Hamburg, Germany).

[0132] CO₂ culture stove Napco digital 6100 (National Appliance Co.,Portland, USA).

[0133] Sterile filters of 22 μm Millex-GS (Millipore, Molshein, France).

[0134] Olympus CHS-2 microscope (Olympus, Tokyo, Japan).

[0135] Glass pipettes of 1, 5 and 10 ml, sterile.

[0136] Piptman P Adjustable volume pipettes of 20, 200, 1000 and 5000 μl(Gilson, France).

[0137] Sterile dishes with 96 flat-bottom wells (Costar, Cambridge,Mass., USA).

[0138] Glass slides and cover slips (Hirschman, Germany).

[0139] Virgin propylene tips, sterile (Daslab, Madrid, Spain).

[0140] Eppendorf sterile tips (Hamburg, Germany).

[0141] Sterile plastic tubes of 5, 10, 15 and 50 ml (Daslab, Madrid,Spain).

[0142] Non-sterile plastic tubes of 3 ml (Indubages, S.A., Manresa,Spain).

[0143] Test:

[0144] Tripan blue (Flucke A G., Buchs S G., Germany).

[0145] Heparin Leo (Lab. Leo, Madrid, Spain).

[0146] Lymphoprep (Ficoll-Hypaque) (Nyegaard Co, Oslo, Norway).

[0147] Physiological saline solution (PSS) Simple chlorated apiroserum(Ibys, Madrid, Spain).

[0148] Opticint “hisafe” scintillation solution (FSA, Leics, UnitedKingdom).

[0149] Foetal calf serum (FCS) (Gibco, Grand Island, N.Y., USA).

[0150] Tritium methyl-³H thymidine. Specific activity 1 μCi/ml.(Amersham inter., United Kingdom).

[0151] Mitogens: Phytohaemaglutinin M (PHA) (Difco Lab., Detroit, Mich.,USA), Concanavalin A (Con A, 2 μg/ml, Sigma Chemical Co., Mich., USA),Immobilised anti-CD3 (OKT3, 5 μg/ml, Ortho-mune, Orthodiagnostic System)and rhIL-2 (100 IU/ml, Hoffman-La Roche, NJ, USA).

[0152] All the reagents included in these tests were diluted in RPMI1640 culture medium (Whitaker Bioproducts, Walkersville, USA)supplemented with 10% of decomplemented foetal calf serum (Biochrom K G,Berlin), L-glutamin (2 mM, Biochrom K G), Hepes (25 mM, Biochrom K G)and antibiotic (1% penicillin streptomycin, Difco Lab, Detroit, Mich.,USA).

[0153] Also, all the mitogens were optimised in a dose response way inorder to obtaining the maximum proliferative response after 5 days ofculture.

[0154] Method:

[0155] Processing of the samples:

[0156] Venous blood: The CMSP were obtained from venous blood extractedby antecubital venous puncture. 50 ml of blood were extracted to whichwere added 50 U of calcic heparin and the mixture was diluted 1/1(vol/vol) with PSS.

[0157] Human mononucleated cells: In order to isolate the CMSP they wereproceeded to be separated from the rest of the blood components by meansof the formation of a density gradient on Ficoll. The cells thusobtained are resuspended in PSS and centrifuged at 400×g for 10 minutes(washing process) and then resuspended in PSS. This operation isrepeated three times. In the last of them, PSS is replaced with completemedium.

[0158] In all the cell suspensions a determination was made of the cellconcentration and the viability by means of dilution with 0.1% TripanBlue and microscope counting in a Neubauer chamber. The percentage oflive cells was established by the exclusion capacity of the colorant.

[0159] General culture conditions: All cell cultures were developedunder conditions of sterility in a vertical laminar flow chamber, usingsingle-use sterile materials or materials sterilised in an autoclave orwith ethylene oxide. The cultures were conserved in a stove kept at atemperature of 37° C., in an atmosphere with 5% CO₂ and relativehumidity of 95%. In the various experiments performed, the purified CMSPcell preparations were incubated in sterile dishes with 96 wells atconcentrations of 5×10⁴ cells/well (200 μl) in the presence of differentconcentrations of different mitogens for 5 days.

[0160] Test:

[0161] The method used for quantifying the cell proliferation wasanalysis of the incorporation of ³H-thymidine (³H-T) into DNAsynthesised de novo, detecting the emission of β radiation from the dryextracts of cell cultures to which the tritiated base had been addedbefore its finalisation and collection. The synthesis of DNA was done intriplicate on sterile dishes with 96 flat-bottom wells.

[0162] From 20 to 24 hour prior to terminating the cell culture, 1 μCiof ³H-T was added to each well containing medium; the cultures weregathered by suction through a glass filter, using a Skatron culturecollector for this.

[0163] The synthesis of DNA has been expressed in counts per minute(cpm). Each test was conducted in triplicate, with those data having avariability greater than 10% compared to the mean of the triplicatebeing rejected, since they could indicate a technical error orcontamination in the culture. The cultures were carried out at aconstant cell volume per well, as well as at a constant volume of 200μl.

[0164] Controls:

[0165] Negative control: cells in the presence of complete culturemedium.

[0166] Positive control: stimulation of cells without the presence oflactone.

[0167] Tests on Human Peripheral Blood Mononuclear Cells. Results:

[0168] The effect was studied of inhibition on the mitogenic activity ofhuman lymphocytes, induced by different mitogenic agents, and of cellactivation, acting alone and/or in combination (Table 2).

[0169] The spontaneous blastogen response of the CMSP falls by 50% if0.66 μg/ml of lactone are added to the culture.

[0170] The blastogen response induced in CMSP due to phytohaemaglutininM (PHA) falls by 50% if 0.25 μg/ml of lactone are added to the culture.

[0171] The blastogen response induced in CMSP by Concavalin A (Con A)falls by 50% if 0.5 μg/ml of lactone are added to the culture.

[0172] The blastogen response induced in CMSP by immobilised anti-CD3(aCD3), falls by 50% if 0.06 μg/ml of lactone are added to the culture.

[0173] The blastogen response induced in CMSP by aCD3+PHA falls by 50%if 0.24 μg/ml of lactone are added to the culture.

[0174] The blastogen response induced in CMSP by aCD3+Con A falls by 50%if 0.21 μg/ml of lactone are added to the culture. TABLE 2 Proliferativeresponse of peripheral blood mononuclear cells. Proliferation of humanperipheral blood mononuclear cells Mitogens (cpm ± DS) Lactone PHA Con AaCD3 aCD3 + aCD3 + μg/ml Medium 10 μg/ml 2 μg/ml 5 μg/ml PHA Con A 100 104 ± 26  1086 ± 308 204 ± 43  39 ± 10 13 ± 4     8 ± 6 10  72 ± 72  71± 14 48 ± 7 119 ± 7 23 ± 7     11 ± 1 1  1805 ± 1870  25118 ± 6642 13294± 3395  1286 ± 255 8689 ± 5361    829 ± 484 0.01  2867 ± 1527  71092 ±3555 55503 ± 9023 40378 ± 219 67011 ± 8270     93139 ± 3070 0.0001  3175± 1574  76612 ± 15020 75329 ± 9144  44721 ± 4082 105395 ± 375     115090± 4133 0 2064 ± 39 77189 ± 686 73639 ± 9993  34088 ± 9498 95158 ± 7752    87804 ± 14148

[0175] Afterwards, a study is made of the effect of inhibition on themitogenic activity in human lymphocytes, induced by different mitogenicagents, and of cell activation, acting alone and/or in combinationfollowing the addition of exogen interleukin 2 (Table 3).

[0176] The blastogen response induced in CMSP by rhIL-2 falls by 50% if0.26 μg/ml of lactone are added to the culture.

[0177] The blastogen response induced in CMSP by PHA+rhIL-2 falls by 50%if 0.29 μg/ml of lactone are added to the culture.

[0178] The blastogen response induced in CMSP by Con A+rhIL-2 falls by50% if 0.27 μg/ml of lactone are added to the culture.

[0179] The blastogen response induced in CMSP by immobilised aCD3+rhIL-2falls by 50% if 0.24 μg/ml of lactone are added to the culture.

[0180] The blastogen response induced in CMSP by immobilisedaCD3+PHA+rhIL-2 falls by 50% if 0.26 μg/ml of lactone are added to theculture.

[0181] The blastogen response induced in CMSP by immobilised aCD3+ConA+rhIL-2 falls by 50% if 0.25 μg/ml of lactone are added to the culture.TABLE 3 Proliferative response of peripheral blood mononuclear cells inthe presence of rhIL-2. Proliferation of human peripheral bloodmononuclear cells Mitogens in the presence of rhIL-2 (100 UI/ml) (cpm ±DS) Lactone PHA Con A aCD3 aCD3 + aCD3 + μg/ml Medium 10 μg/ml 2 μg/ml 5μg/ml PHA Con A 100 918 ± 1    892 ± 199   532 ± 109  400 ± 44 286 ±10    237 ± 109 10 866 ± 66   579 ± 1033  611 ± 75  489 ± 25 304 ± 35  164 ± 65 1 9548 ± 808 110507 ± 698  71210 ± 5211 36280 ± 77 88220 ±3174   66429 ± 9241 0.01 42568 ± 8631 106754 ± 698 54196 ± 80  39771 ±3258 94165 ± 15647  130949 ± 16623 0.0001 56451 ± 4965  130524 ± 8929  96972 ± 18274  87091 ± 7078 133621 ± 12440   154981 ± 15723 0 45902 ±7074  123500 ± 3682  118716 ± 4052  112214 ± 15621 147591 ± 16848  146999 ± 12727

[0182] TABLE 4 Methodological control over the observed effect oflactone towards different mitogens. Proliferation of human peripheralblood mononuclear cells Mitogens (cpm ± DS) Lactone PHA μg/ml Medium 10μg/ml PHA + rhIL-2 100  47 ± 5 906 ± 251   341 ± 21 0.25 283 ± 9 60428 ±5820  144693 ± 9470 0  318 ± 37 113032 ± 1649   158221 ± 6856

[0183] Stability Studies

[0184] Finally, we analyse the stability over time of the productdissolved in dimethylsulphoxide (DMSO) by means of new experiments inwhich, once again, the antiproliferative biological activity of lactoneis demonstrated (Table 4).

[0185] The sample remains for one year at room temperature and at aconcentration of 40.0 mg/ml in DMSO.

1. Derivatives of the compound,

characterised in that they have the following general formula:

where n can take the values 1,2,3; R can be H or CH₃ and R₁ can be CH₃or H.
 2. Compounds according to claim 1 presenting any of the possiblecombinations among the different values of n and the structurescorresponding to the radicals R and R₁.
 3. Derivatives of the compound,

characterised in that they have the following general formula:

where n can take the values 1,2,3; R can be H or CH₃ and R₁ can be CH₃or H.
 4. Compounds according to claim 3 presenting any of the possiblecombinations among the different values of n and the structurescorresponding to the radicals R and R₁.
 5. Compounds according to claims1 or 3 with pharmacological activity and their application in medicinefor the treatment of disorders of the immunological system.
 6. Compoundsaccording to claims 1 or 3 and their application in pharmacy for theiruse in the preparation of the usual galenic forms including in them theoriginal compounds and pro-drugs such as esters, glycosides, etc.